Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

BACKGROUND: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. RESULTS: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. CONCLUSIONS: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

Effects of age and unaccustomed resistance exercise on mitochondrial transcript and protein abundance in skeletal muscle of men.

Mitochondrial dysfunction may contribute to age-associated muscle atrophy. Previous data has shown that resistance exercise (RE) increases mitochondrial gene expression and enzyme activity in older adults; however, the acute response to RE has not been well characterized. To characterize the acute mitochondrial response to unaccustomed RE, healthy young (21 ± 3 yr) and older (70 ± 4 yr) men performed a unilateral RE bout for the knee extensors. Muscle biopsies were taken at rest and 3, 24, and 48 h following leg press and knee extension exercise. The expression of the mitochondrial transcriptional regulator proliferator-activated receptor γ coactivator 1-α (PGC-1α) mRNA was increased at 3 h postexercise; however, all other mitochondrial variables decreased over the postexercise period, irrespective of age. ND1, ND4, and citrate synthase (CS) mRNA were all lower at 48 h postexercise, along with specific protein subunits of complex II, III, IV, and ATP synthase. Mitochondrial DNA (mtDNA) copy number decreased by 48 h postexercise, and mtDNA deletions were higher in the older adults and remained unaffected by acute exercise. Elevated mitophagy could not explain the reduction in mitochondrial proteins and DNA, because there was no increase in ubiquitinated voltage-dependent anion channel (VDAC) or its association with PTEN-induced putative kinase 1 (Pink1) or Parkin, and elevated p62 content indicated an impairment or reduction in autophagocytic flux. In conclusion, age did not influence the response of specific mitochondrial transcripts, proteins, and DNA to a bout of RE.

The unfolded protein response is triggered following a single, unaccustomed resistance-exercise bout.

Endoplasmic reticulum (ER) stress results from an imbalance between the abundance of synthesized proteins and the folding capacity of the ER. In response, the unfolded protein response (UPR) attempts to restore ER function by attenuating protein synthesis and inducing chaperone expression. Resistance exercise (RE) stimulates protein synthesis; however, a postexercise accumulation of unfolded proteins may activate the UPR. Aging may impair protein folding, and the accumulation of oxidized and misfolded proteins may stimulate the UPR at rest in aged muscle. Eighteen younger (n = 9; 21 ± 3 yr) and older (n = 9; 70 ± 4 yr) untrained men completed a single, unilateral bout of RE using the knee extensors (four sets of 10 repetitions at 75% of one repetition maximum on the leg press and leg extension) to determine whether the UPR is increased in resting, aged muscle and whether RE stimulates the UPR. Muscle biopsies were taken from the nonexercised and exercised vastus lateralis at 3, 24, and 48 h postexercise. Age did not affect any of the proteins and transcripts related to the UPR. Glucose-regulated protein 78 (GRP78) and protein kinase R-like ER protein kinase (PERK) proteins were increased at 48 h postexercise, whereas inositol-requiring enzyme 1 alpha (IRE1α) was elevated at 24 h and 48 h. Despite elevated protein, GRP78 and PERK mRNA was unchanged; however, IRE1α mRNA was increased at 24 h postexercise. Activating transcription factor 6 (ATF6) mRNA increased at 24 h and 48 h, whereas ATF4, CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage protein 34 mRNA were unchanged. These data suggest that RE activates specific pathways of the UPR (ATF6/IRE1α), whereas PERK/eukaryotic initiation factor 2 alpha/CHOP does not. In conclusion, acute RE results in UPR activation, irrespective of age.

Elevated mitochondrial oxidative stress impairs metabolic adaptations to exercise in skeletal muscle.

Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 (+/-) mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 (+/-) mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 (+/-) mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.

Oxidative stress and Nrf2 signaling in McArdle disease.

McArdle disease (MD) is a metabolic myopathy due to myophosphorylase deficiency, which leads to a severe limitation in the rate of adenosine triphosphate (ATP) resynthesis. Compensatory flux through the myoadenylate deaminase > > xanthine oxidase pathway should result in higher oxidative stress in skeletal muscle; however, oxidative stress and nuclear factor erythroid 2-related factor 2 (Nrf2) mediated antioxidant response cascade in MD patients have not yet been examined. We show that MD patients have elevated muscle protein carbonyls and 4-hydroxynonenal (4-HNE) in comparison with healthy, age and activity matched controls (P < 0.05). Nuclear abundance of Nrf2 and Nrf2-antioxidant response element (ARE) binding was also higher in MD patients compared with controls (P < 0.05). The expressions of Nrf2 target genes were also higher in MD patients vs. controls. These observations suggest that MD patients experience elevated levels of oxidative stress, and that the Nrf2-mediated antioxidant response cascade is up-regulated in skeletal muscle to compensate.

Monocarboxylate transporters and mitochondrial creatine kinase protein content in McArdle disease.

McArdle disease (MD) is a metabolic myopathy due to myophosphorylase deficiency. We examined monocarboxylate transporters (MCT) and creatine kinase (CK) protein content in skeletal muscle from MD patients and age-matched controls to evaluate potential cellular adaptations that compensate for the loss of glycogenolysis. Our findings of higher MCT1 and mitochondrial CK suggest that proteins related to extra-muscular fuel uptake and intra-muscular energy transduction are up-regulated without change in mitochondrial mass in MD patients.

Elevated SOCS3 and altered IL-6 signaling is associated with age-related human muscle stem cell dysfunction.

Aging is associated with increased circulating interleukin-6 (IL-6) and a reduced myogenic capacity, marked by reduced muscle stem cell [satellite cell (SC)] activity. Although IL-6 is important for normal SC function, it is unclear whether elevated IL-6 associated with aging alters SC function. We hypothesized that mild chronically elevated IL-6 would be associated with a blunted SC response through altered IL-6 signaling and elevated suppressor of cytokine signaling-3 (SOCS3) in the elderly. Nine healthy older adult men (OA; 69.6 ± 3.9 yr) and 9 young male controls (YC; 21. 3 ± 3.1 yr) completed 4 sets of 10 repetitions of unilateral leg press and knee extension (75% of 1-RM). Muscle biopsies and blood were obtained before and 3, 24, and 48 h after exercise. Basal SC number was 33% lower in OA vs. YC, and the response was blunted in OA. IL-6(+)/Pax7(+) cells demonstrated a divergent response in OA, with YC increasing to 69% at 3 h and peaking at 24 h (72%), while IL-6(+)/Pax7(+) cells were not increased until 48 h in OA (61%). Type II fiber-associated phosphorylated signal transducer and activator of transcription (pSTAT3)(+)/Pax7(+) cells demonstrated a similar delay in OA, not increasing until 48 h (vs. 3 h in YC). SOCS3 protein was 86% higher in OA. These data demonstrate an age-related impairment in normal SC function that appears to be influenced by SOCS3 protein and delayed induction of IL-6 and pSTAT3 in the SCs of OA. Collectively, these data suggest dysregulated IL-6 signaling as a consequence of aging contributes to the blunted muscle stem cell response.

Myostatin is associated with age-related human muscle stem cell dysfunction.

Human aging is accompanied by a progressive loss of muscle mass (sarcopenia). We tested the hypothesis that older males (OMs, 70±4 yr, n=9) would have a blunted myogenic response to a physiological stimulus compared to younger controls (21±3 yr, n=9). Subjects completed an acute bout of intense unilateral muscle loading. Young healthy males matched for body mass and activity level served as the control group. Muscle biopsies and blood were obtained before and at 3, 24, and 48 h after muscle loading. The muscle stem cell response was analyzed using flow cytometry, immunofluorescent microscopy, and standard protein and mRNA analysis. OMs had 35% fewer basal stem cells and a type II fiber-specific impairment in stem cell content and proliferation. Myogenic determination factor staining and cell cycle analysis illustrated a severely blunted progression through the myogenic program. Myostatin protein and mRNA were 2-fold higher in OMs. Stem cell-specific myostatin levels were not different at baseline; however, there were 67% more myostatin-positive type II-associated stem cells in OMs at 24 h. These data illustrate an age-related impairment of stem cell function in a fiber type-specific manner. The greater colocalization of myostatin with stem cells provides a mechanism for the impaired myogenic capacity of aged muscle.

Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage.

Massage therapy is commonly used during physical rehabilitation of skeletal muscle to ameliorate pain and promote recovery from injury. Although there is evidence that massage may relieve pain in injured muscle, how massage affects cellular function remains unknown. To assess the effects of massage, we administered either massage therapy or no treatment to separate quadriceps of 11 young male participants after exercise-induced muscle damage. Muscle biopsies were acquired from the quadriceps (vastus lateralis) at baseline, immediately after 10 min of massage treatment, and after a 2.5-hour period of recovery. We found that massage activated the mechanotransduction signaling pathways focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2), potentiated mitochondrial biogenesis signaling [nuclear peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)], and mitigated the rise in nuclear factor κB (NFκB) (p65) nuclear accumulation caused by exercise-induced muscle trauma. Moreover, despite having no effect on muscle metabolites (glycogen, lactate), massage attenuated the production of the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and reduced heat shock protein 27 (HSP27) phosphorylation, thereby mitigating cellular stress resulting from myofiber injury. In summary, when administered to skeletal muscle that has been acutely damaged through exercise, massage therapy appears to be clinically beneficial by reducing inflammation and promoting mitochondrial biogenesis.

Effects of creatine and exercise on skeletal muscle of FRG1-transgenic mice.

BACKGROUND: The FRG1-transgenic mouse displays muscle dysfunction and atrophy reminiscent of fascioscapulohumeral muscular dystrophy (FSHD) and could provide a model to determine potential therapeutic interventions. METHODS: To determine if FRG1 mice benefit from treatments that improve muscle mass and function, mice were treated with creatine alone (Cr) or in combination with treadmill exercise (CrEX). RESULTS: The CrEx treatment increased quadriceps weight, mitochondrial content (cytochome c oxidase (COX) activity, COX subunit one and four protein), and induced greater improvements in grip strength and rotarod fall speed. While Cr increased COX subunits one and four protein, no effect on muscle mass or performance was found. Since Cr resulted in no functional improvements, the benefits of CrEx may be mediated by exercise; however, the potential synergistic action of the combined treatment cannot be excluded. CONCLUSION: Treatment with CrEx attenuates atrophy and muscle dysfunction associated with FRG1 overexpression. These data suggest exercise and creatine supplementation may benefit individuals with FSHD.

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice.

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.

Effects of exercise and muscle type on BDNF, NT-4/5, and TrKB expression in skeletal muscle.

Muscle-derived neurotrophins are thought to contribute to the adaptation of skeletal muscle to exercise, but the effects of brief exercise interventions on BDNF, NT-4/5, and trkB are not understood. RNA was extracted for RT-PCR from soleus and medial gastrocnemius of Sprague-Dawley rats exercised on a treadmill at speeds up to 20 m/min at 5% incline for 5 or 10 days. BDNF expression was elevated in soleus following 5 days (184%, P < 0.001) but not 10 days of exercise. NT-4/5 and trkB were not affected at either time-point. BDNF mRNA was significantly higher in soleus at rest when compared with medial gastrocnemius (193%, P < 0.05). No significant effects of muscle type were detected for NT-4/5 and trkB. Our results indicate differential control of BDNF expression between soleus and medial gastrocnemius following 5 days of exercise. BDNF may be a protein with an uncharacterized contribution to the acute adaptation of skeletal muscle to exercise, whereas NT-4/5 shows no response.